Nicotinic receptor agonists for the treatment of inflammatory diseases

ABSTRACT

This invention relates to the use of nicotine receptor agonists for treating inflammatory diseases, including a variety of pulmonary diseases. Such agonists have fewer side effects than other anti-inflammatory drugs, such as steroids. Moreover, these agonists can be used alone or in combination with other anti-inflammatory drugs to alleviate pulmonary diseases.

FIELD OF THE INVENTION

The present invention relates to the treatment of inflammatory diseases,including a variety of pulmonary diseases, through the use oradministration of nicotinic receptor agonists.

BACKGROUND OF THE INVENTION

Although we breathe more than one cubic meter of air every hour, ourlung defense mechanisms usually deal with the large quantities ofparticles, antigens, infectious agents and toxic gases and fumes thatare present in inhaled air. The interaction of these particles with theimmune system and other lung defense mechanisms results in thegeneration of a controlled inflammatory response which is usuallyprotective and beneficial. In general, this process regulates itself inorder to preserve the integrity of the airway and alveolar epithelialsurfaces where gas exchange occurs. In some cases, however, theinflammatory response cannot be regulated and the potential for tissueinjury is increased. Depending on the type of environmental exposure,genetic predisposition, and a variety of ill-defined factors, abnormallylarge numbers of inflammatory cells can be recruited at different sitesof the respiratory system, resulting in illness or disease.

The inflammatory response to inhaled or intrinsic stimuli ischaracterized by a non-specific increase in the vascular permeability,the release of inflammatory and chemotactic mediators includinghistamine, eicosanoids, prostaglandins, cytokines and chemokines. Thesemediators modulate the expression and engagement ofleukocyte-endothelium cell adhesion molecules allowing the recruitmentof inflammatory cells present in blood.

A more specific inflammatory reaction involves the recognition and themounting of an exacerbated, specific immune response to inhaledantigens. This reaction is involved in the development of asthma,Hypersensitivity pneumonitis (HP) and possibly sarcoidosis.Dysregulation in the repair mechanisms following lung injury maycontribute to fibrosis and loss of function in asthma, pulmonaryfibrosis, chronic obstructive pulmonary disease (COPD), and chronic HP.

It was previously reported that the incidence of HP is much lower amongcurrent smokers than in non-smokers (1-4). Sarcoidosis is also lessfrequent in smokers than in non smokers (5, 6). The mechanismsunderlying the beneficial effects of cigarette smoking on thedevelopment of HP and other inflammatory diseases are still unknown butmay be linked to the immunomodulatory effect of nicotine. There areclinical observations of asthma de novo or exacerbation after smokingcessation. Proof of this is difficult to obtain and any protectiveeffects of nicotine in the prevention or treatment of asthma are likelyoverwhelmed by the negative effects of tobacco smoke with its thousandsof constituents.

The protective effect of smoking has also been reported in otherdiseases, the most studied being ulcerative colitis, an inflammatoryintestinal disease (7, 8). Nicotine has been successfully used in thetreatment of this disease (9, 10). Other studies have looked at thepossible therapeutic value of nicotine in the treatment of Alzheimer'sdisease and Parkinson's disease (11, 12).

Nicotinic receptors are pentamers made up of five polypeptide subunitswhich act as ligand-gated ions channels. When the ligand binds to thereceptor, a conformational change in the polypeptide occurs, opening acentral channel that allows sodium ion to move from the extracellularfluid into the cytoplasm. Four types of subunits have been identified:α, β, γ and δ. The receptor can consist of any combination of these fourtypes of subunits (13). Recent work has shown that alveolar macrophages(AM) can express the α-7 subunit (14), while bronchial epithelial cellsexpress the α-3, α-5 and α-7 subunits (15), and lymphocytes the α-2,α-5, α-7, β-2 and β-4 subunits (14). Fibroblasts (16) and airway smoothmuscles cells (17) also express these receptors. Therefore, residentpulmonary cells (AM, dendritic cells, epithelial cells, fibroblasts,etc.) and those recruited in inflammatory diseases (lymphocytes,polymorphonuclear cells) express nicotinic receptors.

Nicotinic receptor activation in lymphocytes affects the intracellularsignalization, leading to incomplete activation of the cell. In fact,nicotine treatment upregulates protein kinase activity, which in turnupregulates phospholipase A2 (PLA2) activity. PLA2 is responsible forcleaving phosphoinositol-2-phosphate (PIP2) into inositol-3-phosphate(IP3) and diacylglycerol (DAG) (18, 19). The continuous presence of IP3in the cell would appear to result in the desensitization of calciumstores, leading to their depletion (19). This observation could explainthe fact that nicotine-treated lymphocytes do not release enough calciuminto the cytoplasm to activate transcription factors such as NFk-B (20).

Nicotine, the major pharmacological component of cigarette smoke, is oneof the best known nicotinic receptor agonists (21). This naturalsubstance has well defined anti-inflammatory and immunosuppressiveproperties (22), and may have anti-fibrotic properties (23). Exposure ofanimals to smoke from cigarettes with high levels of nicotine is moreimmunosuppressive than that from low-nicotine cigarettes (24). Moreover,treatment of rats with nicotine inhibits the specific antibody responseto antigens and induces T cell anergy (25). Although they are increasedin number, AM from smokers show a decreased ability to secreteinflammatory cytokines in response to endotoxins ((20, 25, 26)) andnicotine seems to be the responsible component of this inhibition (26).One study also showed that peripheral blood lymphocytes from smokersexpress higher levels of FAS ligand (FASL) and that nicotine increasesFASL expression on lymphocytes from non-smokers, indicating thatnicotine may affect cell apoptosis (27). Nicotine was also shown to havean inhibitory effect on the proliferation and extracellular matrixproduction of human gingival fibroblasts in vitro (23). Of interest,nicotine treatment seems to up-regulate the expression of nicotinicreceptors (28).

Nicotinic agonists may down-regulate T cell activation, indeed, nicotinehas been shown to affect T cell expression of the co-stimulatorymolecules CD28 and CTLA4 (29).

The B7/CD28/CTLA4 co-stimulatory pathway plays a key regulatory role inT-cell activation and homeostasis (30, 31). Two signaling pathways areinvolved. A positive signal involves the engagement of B7 (CD80/CD86)molecules with T cell CD28 receptors which results in the potentiationof T cell responses (proliferation, activation, cytokine expression, andsurvival) (32). A negative signal involves B7 interactions with CTLA4 onactivated T cells, leading to a downmodulation of T cell responses (33,34). The balance between CD28 and CTLA4 derived signals may alter theoutcome of T-cell activation.

In HP, it was previously reported that an upregulation of B7 moleculeexpression on AM in patients with active HP (35) and in murine HP (36).It was also shown that a blockade of the B7-CD28 co-stimulatory pathwayin mice inhibited lung inflammation (36). These results alsodemonstrated that the expression of B7 molecules on AM is lower insmokers than in non-smokers and that an in vitro influenza virusinfection is able to upregulate B7 expression in normal human AM but notin AM from smokers; whether this is due to nicotine or other substancespresent in cigarette smoke is unknown (35). An up-regulation of the B7molecules has also been reported in asthma (37, 38) and sarcoidosis(39).

Epibatidine is the most potent nicotinic agonist known so far (40). Ithas anti-inflammatory and analgesic properties. In fact, its analgesicpotential is two hundred times that of morphine (40). This molecule isalso known to inhibit lymphocyte proliferation in vitro (41). Thebinding of epibatidine to the receptor is non-specific (42).Unfortunately, epibatidine has major toxic side effects mostly on thecardiovascular and the central nervous systems making it inappropriatefor use as an anti-inflammatory drug to treat pulmonary diseases (40).

Dimethylphenylpiperazinium (DMPP) is a synthetic nicotinic agonist thatis non-specific (13). Its potency for the receptor is about the same asnicotine, depending on the kind of cells implicated in the stimulation(43). Its advantage over nicotine and other nicotinic agonists is thatits chemical configuration prevents it from crossing the blood-brainbarrier, thus causing no addiction or other central nervous effects(13). The anti-inflammatory properties of DMPP are not well described.However, it has been shown that a chronic in vivo treatment coulddecrease the number of white blood cells, decrease the cytokineproduction by splenocytes and decrease the activity of natural killercells (44). The effect of DMPP on airway smooth muscle cells has alsobeen tested. DMPP has an initial short contractive effect which isfollowed by a relaxing effect when the cells are in contact with theagonist for a longer period of time (45). This bronchodilatory effectwould not in itself make DMPP a potentially useful treatment of asthma,since more potent bronchodilators are currently available on the market(B2 agonists). However, the properties of this nicotinic receptoragonist are important since this drug could be safely administered toasthmatics and COPD patients for its anti-inflammatories properties.Moreover, there is no evidence that DMPP has any toxic effect on majororgans such as the heart, the brain, the liver or the lungs.

Despite advances in the treatment of inflammatory illnesses, includingpulmonary inflammatory diseases, treatment using available drugs oragents frequently results in undesirable side effects. For example, theinflammation of COPD is apparently resistant to corticosteroids, andconsequently the need for the development of new anti-inflammatory drugsto treat this condition has been recognized (46).

Similarly, while corticosteroids and other immunosuppressive medicationshave been routinely employed to treat pulmonary fibrosis, they havedemonstrated only marginal efficacy (47).

There is thus a need for new and reliable methods of treatinginflammatory diseases, including pulmonary inflammatory diseases, in amanner that alleviates their symptoms without causing side effects.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novelmethod for treating inflammatory diseases. Specifically, a novel methodis described for treating pulmonary inflammatory diseases through theuse or administration of nicotinic receptor agonists.

The idea of using nicotine or other nicotinic receptor agonists to treatinflammatory pulmonary disease is novel. Despite the impressiveanti-inflammatory and immunosuppressive properties of nicotine and othernicotinic receptor agonists, their usefulness in the treatment ofallergic and other inflammatory lung diseases has not previously beendisclosed. Nicotine itself is a safe substance that does not seem tohave any long term side effects (48,49). Smoke-related diseases of thelungs, heart and arteries are not caused by nicotine but by thethousands of other chemicals present in the inhaled smoke. The mainproblem is that nicotine crosses the blood-brain barrier, inducingaddiction. These are major reasons for the lack of prior interest innicotinic agonists in the treatment of lung diseases. The harmfuleffects of cigarette smoking are obvious. Although nicotine is notresponsible for the toxic effects of cigarette smoking (49), theassociation remains.

The present invention thus proposes the use nicotinic receptor agonists,such as DMPP, to treat inflammatory lung diseases such as asthma, COPD,interstitial pulmonary fibrosis (IPF), sarcoidosis, HP, andbronchiolitis obliterans with organizing pneumonitis (BOOP). The drugcould be administered orally, or preferably by targeted deliverydirectly to the lung by aerosolisation with different and preferredvehicules thus minimizing any systemic effects.

The anti-inflammatory and immunosuppressive properties, as well asminimal side effects, of nicotinic receptor agonists make these drugsideally suited for medical use in the treatment of a large variety oflung diseases that are characterized by bronchial or interstitialinflammation. These diseases include diseases such as asthma, COPD, IPF,sarcoidosis, HP and BOOP.

Other objects, advantages and features of the present invention willbecome more apparent upon reading the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Total and differential cell counts in BAL cells. There was amarked inhibition of total cell counts in nicotine treated mice duemainly to a decrease in the lymphocyte population.

FIG. 2: IFN-γ mRNA expression in isolated lung mononuclear cells. Asignificant inhibition of IFN-γ mRNA was observed.

FIG. 3: TNF-a mRNA expression was induced by a 24 h Lipopolysaccharidescomponent of gram negative cell walls (LPS) stimulation. Results areexpressed as a % of expression, 100% being attributed to the LPS alonegroup. The intensity of the band was obtained by dividing the intensityof the TNF-α band by that of β-actin. Treatment of stimulated cells withdifferent doses (40 to 160 uM for nicotine and DMPP) induced a drop ofTNF-α mRNA expression. The greatest effect was obtained with the 40 uMconcentration of nicotine (a 98% reduction of expression), while alldoses of DMPP caused a 60 to 50% reduction of expression.

FIG. 4: TNF-α mRNA expression was induced by a 24 h saccharopolysporarectivirgula (SR) stimulation. Results are expressed as described inFIG. 5. Treatment of stimulated cells with different doses (80 and 160μM for nicotine and 40 to 160 μM for DMPP) induced a down-regulation ofTNF-α mRNA expression. Only the 160 μM dose of nicotine had an effect onmRNA expression, while the 40 and 80 μM doses of DMPP induced up to 60%of reduction of TNF-α mRNA expression.

FIG. 5: IL-10 mRNA expression was induced by a 24 h LPS stimulation.Results are expressed as described in FIG. 3. Treatment of stimulatedcells with different doses (40 to 160 μM for both nicotine and DMPP)induced a down-regulation of IL-10 mRNA expression. The largest drop ofexpression (a 87% reduction) occurred with 40 uM nicotine. DMPP induceda 55 to 40% reduction of expression for all three doses.

FIG. 6: IL-10 mRNA expression was induced by a 24 h SR stimulation.Treatment of stimulated cells with different doses (80 and 160 μM fornicotine and 40 to 80 μM for DMPP) induced a down-regulation of IL-10mRNA expression. The greatest drop in mRNA expression with the nicotinetreatment occurred at 160 μM (60% drop of expression), and at 80 μM (90%drop of expression) with the DMPP treatment.

FIG. 7: IFN-γ mRNA expression was induced in RAW 264.7 cells by a 24 hLPS stimulation. Results are expressed as described in FIG. 3. Treatmentof stimulated cells with different doses of DMPP induced a reduction inIFN-γ mRNA expression. The largest drop of expression (a 80% reduction)occurred with 40 μM DMPP.

FIG. 8: a) The expression of CD 80 was induced with either LPS (38%) orSR antigen (35%). Nicotine treatment (40 μM for 48 h) reduced theexpression to 20% in LPS stimulated cells and 26% in SR stimulatedcells. b) The expression of CD 80 was induced with either LPS (38%) orSR antigen (35%). DMPP treatment (40 μM for 48 h) reduced the expressionto 17% in LPS stimulated cells and 20% in SR stimulated cells.

FIG. 9: IFN-γ mRNA expression in T lymphocytes isolated from BALperformed on HP patients. DMPP treatment reduced expression of IFN-γ inthese cells.

FIG. 10: CD 86 expression in total cells from a BAL that was performedon a normal subject. Cells that were treated with DMPP express 50% lessCD 86 than non-treated cells.

FIG. 11: BAL cells from DMPP, nicotine and epibatidine treated mice.Treatment with nicotine and epibatidine had a significant inhibitoryeffect on SR-induced inflammation after 24 hours.

FIG. 12: A significant inhibitory effect of DMPP on lung inflammationwas found when we increased the number of animals.

FIG. 13: TNF levels in BAL fluid (BALF) from DMP-treated mice. DMPPdecreased significantly BALF TNF levels.

FIG. 14: Effect of intra-peritoneal treatment with increasing doses ofDMPP on total cell accumulation in BAL of asthmatic mice. The number ofcells was highly elevated in OVA challenged and non-treated mice. TheDMPP treatment significantly reduced cell counts at the 0.5 and 2.0mg/kg doses.

FIG. 15: Differential counts for the dose response. The OVA challengedmice (OVA OVA) had more eoosinophils and lymphocytes in their BALcompared to the control group (sal sal). The DMPP treatmentsignificantly reduced the presence of both osinophils and lymphocytes inBAL in all groups (n=8; p<0.05).

FIG. 16: Second dose response for the DMPP IP treatment effect on totalcell accumulation in BAL of asthmatic mice. The number of cells washighly elevated in OVA challenged and non-treated mice. The DMPPtreatment significantly reduced total cells at the 0.1 and 0.5 mg/kgdoses.

FIG. 17: Differential counts from the second dose response. The DMPPtreatment significantly reduced eosinophil and lymphocyte counts in the0.1 and 0.5 mg/kg doses, 0.5 mg/kg being the most effective dose for theanti-inflammatory effect of DMPP.

FIG. 18: BAL IL-5 levels from control, asthmatic and treated mice. TheOVA challenges increased IL-5 levels in BAL, while the DMPP treatmenthad a significant inhibitory effect on IL-5 levels in the 0.5 mg/kgtreated-group of mice.

FIG. 19: Lung resistance after metacholine challenges from normal,asthmatic and asthmatic treated with 0.5 mg/kg intranasal DMPP. DMPPreduces the % of augmentation of lung resistance compared to asthmaticmice.

FIG. 20: The provocative challenge dose of 200% lung resistanceaugmentation (PC 200) was calculated. DMPP significantly reduced thePC200 in treated-mice compared to asthmatic mice (p=0.04; n=6).

FIG. 21 :IL-4 mRNA expression was induced by a 24 h LPS stimulation.Results are expressed as described in FIG. 3. Cells were treated withdifferent doses (40 to 160 μM for both nicotine and DMPP). The nicotinetreatment induced a drop in the IL-4 mRNA expression (up to a 90%reduction of expression in the 40 μM group). DMPP treatment completelyblocked IL-4 mRNA expression in the LPS stimulated cells, at all doses.

FIG. 22: Effect of DMPP on blood eosinophil transmigration. DMPP inducesa dose-related inhibition of eosinophil transmigration across anartificial basement membrane.

FIG. 23: Effect of mecamylamine, a nicotinic antagonist, on theinhibitory effect of DMPP on blood eosinophil transmigration.Mecamylamine reverses the effect of DMPP, suggesting that nicotinicreceptor activation is necessary for the DMPP inhibitory effect.

FIG. 24: Effect of other nicotinic agonists on transmigration of bloodeosinophils. Nicotine, epibatidine and cytosine all reduce bloodeosinophil transmigration.

FIG. 25: Effect of DMPP on collagen 1A mRNA expression by normal humanlung fibroblasts. DMPP inhibits collagen 1A mRNA expression in a dosedependant manner.

FIG. 26: Effect of nicotine on collagen 1A mRNA expression by human lungfibroblasts. Nicotine inhibits collagen 1A mRNA expression at 1 and 10μM while the higher doses have no inhibitory effect.

FIG. 27: Effect of epibatidine, another nicotinic agonist, on collagen1A mRNA expression by human lung fibroblasts. Epibatidine also has aninhibitory effect on collagen 1A mRNA expression.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The presence of nicotinic receptors on inflammatory and pulmonary cellshas been described previously. However, the novelty of the presentinvention resides in the observation that nicotinic receptor agonistsappear to be useful in the treatment of inflammatory lung diseases, andin the related discovery of the anti-inflammatory and immunosuppressiveproperties of nicotinic agonists specifically directed againstmechanisms involved in the pathogenesis of such inflammatory pulmonarydiseases as asthma, HP, sarcoidosis, BOOP, IPF, and COPD. An example ofthis is the effect of cigarette smoke on the expression of the B7co-stimulatory molecules.

Two animal models were used to study the effects of nicotinicantagonists in inflammatory pulmonary diseases: an HP model and anasthma model. With both of these models, the effects of nicotinicreceptor agonists (both selective and non-selective) were studied onlung physiology, and inflammation. In vitro studies were performed usingisolated inflammatory cells from the animal studies or from patients aswell as commercially available cell lines in an attempt to understandthe mechanisms by which nicotinic agonists down-regulate inflammation.

Initially, experiments were conducted with non-specific agonists, i.eagonists that bind to all nicotinic receptor subunits (nicotine,dimethylphenylpiperazinium (DMPP) and epibatidine) (13, 42). A β4subunit specific agonist, cytisine (42), was also tested to see whethera specific stimulation could also have anti-inflammatory effects.

For the purposes of the present application, the term “animal” is meantto signify human beings, primates, domestic animals (such as horses,cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, etc.) and othermammals. Generally, this term is used to indicate living creatureshaving highly developed vascular systems.

For the purposes of the present invention, agonists or agents aremolecules or compounds that bind to and modulate the function of thenicotinic receptor. Preferred agents are receptor-specific and do notcross the blood-brain barrier, such as DMPP. Useful agents may be foundwithin numerous chemical classes, though typically they are organiccompounds and preferably, small organic compounds. Small organiccompounds have a molecular weight of more than 150 yet less than about4,500, preferably less than about 1500, more preferably, less than about500. Exemplary classes include peptides, saccharides, steroids,heterocyclics, polycyclics, substituted aromatic compounds, and thelike.

Selected agents may be modified to enhance efficacy, stability,pharmaceutical compatibility, and the like. Structural identification ofan agent may be used to identify, generate, or screen additional agents.For example, where peptide agents are identified, they may be modifiedin a variety of ways as described above, e.g. to enhance theirproteolytic stability. Other methods of stabilization may includeencapsulation, for example, in liposomes, etc. The subject bindingagents are prepared in any convenient way known to those skilled in theart.

For therapeutic uses, agents affecting nicotinic receptor function maybe administered by any convenient means. Small organics are preferablyadministered orally; other compositions and agents are preferablyadministered parenterally, conveniently in a pharmaceutically orphysiologically acceptable carrier, e.g., phosphate buffered saline, orthe like. Typically, the compositions are added to a retainedphysiological fluid such as blood or synovial fluid.

As examples, many such therapeutics are amenable to direct injection orinfusion, topical, intratracheal/nasal administration e.g. throughaerosol, intraocularly, or within/on implants (such as collagen, osmoticpumps, grafts comprising appropriately transformed cells, etc. withtherapeutic peptides. Generally, the amount administered will beempirically determined, typically in the range of about 10 to 1000 μg/kgof the recipient. For peptide agents, the concentration will generallybe in the range of about 50 to 500 μg/ml in the dose administered. Otheradditives may be included, such as stabilizers, bactericides, etc. Theseadditives will be present in conventional amounts.

Nicotinic agonists would not replace all drugs that are currently usedto treat inflammatory lung diseases and the airflow obstruction that isoften associated with these diseases. Bronchodilators remain useful forthe immediate release of bronchospasms. However, bronchodilators have noeffect on the underlying cause or inflammation.

Corticosteroids are potent anti-inflammatory drugs. Their systemic usecauses major side effects that preclude their long-term uses wheneverpossible. Inhaled poorly absorbed steroids are useful to treat airwayinflammation. At low doses these drugs have little or no side effects.However, higher doses increase the risks for oral candidasis, vocalcords paralysis, cataracts and osteoporosis. Inhaled steroids have noeffects on lung interstitium and have no anti-fibrotic properties (57).

More recent drugs, such as anti-leukotrienes, are useful in someasthmatics (58) but have no effects in COPD and other lung diseases.These drugs have anti-inflammatory properties limited to the componentsof inflammation caused by leukotrienes (59). The treatment ofinterstitial lung disease such as IPF, Sarcoidosis, HP, and BOOPbasically rests on the use of systemic corticosteroids. This treatmentis effective in controlling some of the inflammation but unfortunatelyinduces serious side effects and does not reverse underlying fibroticchanges. Immunosuppressive agents such as cyclophosphamide andazathioprine are sometimes tried in severe IPF but their therapeuticvalues are unproven and at most, very limited (60). In essence, lungfibrosis is usually progressive and untreatable, with most IPF patientsdying of this condition (61).

Nicotinic agonists may be useful as a steroid sparing or replacing drug.By targeting their delivery to the lung phagocytes, these drugs could behelpful in controlling both airway and interstitial inflammation. Onemajor advantage of nicotinic agonists over corticosteroids, besideshaving fewer side effects, is the fact that these agonists have a directeffect on fibroblasts and could therefore prevent or reverse fibrosis inthe airways and in the lungs, something corticosteroids cannot do.Interstitial fibrosis is the hallmark if IPF, a major sequel of HP andsarcoidosis, and airway fibrosis is a prevailing finding in chronicasthma (57).

Other substances are actively being studies as potential new treatmentsfor inflammatory lung diseases. Many cytokines are specifically targeted(e.g. IL-5, IL-13, IL-16 . . . ) (62). It is believed that because ofthe complexity of pathways involved in inflammation, any one specificcytokine or other inflammatory mediator is unlikely to have asignificant impact on the treatment of these lung diseases. Nicotinicreceptor agonists, not unlike corticosteroids, have the advantage oftargeting a broad spectrum of the inflammatory response. Therein liestheir potential in the treatment of inflammatory lung diseases.

EXAMPLES

I—Hypersensitivity-like Inflammation

Effect of nicotinic agonists on long term-nnduced hypersensitivitypneumonitis (HP) in Mice.

Example 1 In Vivo HP Studies

The hypothesis is that the stimulation of nicotinic receptors withnicotine down-regulates the immune response to HP antigens viainflammatory cytokine suppression and inhibition of specificantigen-mediated cellular activation.

This model was selected because, as mentioned previously, the incidenceof HP is lower in smokers than in non-smokers (50), and because thismodel is well described. HP was induced by the administration ofSaccharopolyspora rectivirgula (SR) antigen, the causative agent offarmer's lung (51), a form of HP. Mice were simultaneously treated withintra-peritoneal (IP) nicotine, with doses ranging from 0.5 to 2.0mg/kg, twice a day. Nicotine administration significantly reduced thenumber of total cells found in the bronchoalveolar lavage (BAL) of thesemice. The population that was the most affected by nicotine treatmentwere lymphocytes (FIG. 1). Pulmonary macrophages and lymphocytes wereisolated, and stimulated with anti-CD3+recombinant IL-2. The productionof IFN-γ mRNA by these cells, a cytokine known to be involved in thedevelopment of HP and other pulmonary inflammatory diseases (52), wasmeasured. Cells from nicotine treated animals showed significantly lowerexpression of IFN-γ mRNA than cells from non-treated animals (FIG. 2).

Example 2 In Vitro Studies Showing the Effect of Nicotinic Agonists onCytokine Expression

To further clarify the mechanisms involved in suppressive effect ofnicotine in the in vivo model, an alveolar macrophage cell line wasused.

The effect of nicotine or DMPP treatment on AMJ2-C11 cells was tested onTNF-α and IL-10 mRNA expression by RT-PCR. These cytokines are involvedin the development of pulmonary inflammatory diseases such as HP, asthmaand sarcoidosis (52-55). Nicotine and DMPP treatments showed a greatdecrease in TNF mRNA expression (up to a 98% reduction of expression inLipopolysaccharides component of gram negative cell walls (LPS)stimulated and treated with 40 μM nicotine), but not in a dose-dependantmanner (FIG. 3). Similar results were observed with SR-stimulated cells(FIG. 4). This non-dose dependant response can be explained by nicotinicreceptor desensitization due to a large quantity of agonist in themedium. IL-10 mRNA expression was also impaired by nicotine and DMPPtreatment. The best down-regulation occurred at a dosage of 40 μMnicotine (LPS stimulated; 88 % reduction of mRNA expression; FIG. 5) andat a dosage of 80 μM DMPP (SR stimulated ; 87% mRNA expressionreduction; FIG. 6). Once again, the effect was not dose-dependant.

Another macrophage cell line (RAW 264.7, ATCC) was used to test theeffect of DMPP on IFN-γ expression by RT-PCR, because AMJ2-C11 cells didnot appear to express IFN-γ mRNA (data not shown). Cells were stimulatedwith 50 μg/ml of SR antigen and incubated with DMPP at doses rangingfrom 40 to 160 μM. DMPP treatment reduced the expression of INF-γ inthese cells by up to 75% with the 40 μM dose (FIG. 7). Once more, theeffect did not seem to be dose-dependant.

Example 3 In Vitro Effects of Nicotinic Agonists on Co-stimulatoryMolecule Expression

The effects of nicotine and DMPP on B7 (CD80) molecule expression weretested in vitro. AMJ2-C11 cells (mouse alveolar macrophages, from theATCC) were incubated with 40 μM nicotine or DMPP and stimulated with LPS(0.1 μg/ml) or SR antigen (50 μg/ml) for 48 hours. The percentage ofexpression of CD80 in treated cells was about one half of the expressionfound in LPS and SR stimulated non-treated cells (FIGS. 8( a) and (b)).

Example 4 Studies on Human BAL Cells (AM and Lymphocytes)

Since one goal was to treat patients with DMPP or similar drugs, theeffect of this drug was verified on lymphocytes from patients with HP.BAL were performed on patients with HP. Lymphocytes were isolated fromthe other BAL cells, stimulated with PHA and incubated with DMPP. Thedose-response of DMPP were tested on cytokine mRNA production (byRT-PCR) for IFN-γ (FIG. 9).

A broncho-alveolar lavage was performed on a normal subject, andalveolar macrophages were isolated. SR-stimulated and nicotine or DMPPtreated cells showed once again about half of the expression of CD86than non-treated cells (FIG. 10).

Example 5 Investigation of the Effect of Other Nicotinic Agonists on theShort Term SR-induced Acute Inflammation

The intranasal instillation of Saccharopolyspora rectivirgula (SR)antigens, the causative agent for farmer's lung, to mice, induces aprominent inflammatory response in the lung. Neutrophils are the firstinflammatory cells recruited at the site of inflammation. Treatment ofmice with DMPP (0.5 mg/kg), nicotine (0.5 mg/kg) and epibatidine (2μg/kg) had a marked inhibitory effect on SR-induced inflammation (FIG.11). Nicotinic agonists were administered intra-nasally in 50 μl volumeevery 6 h and mice were sacrificed 24 hr after SR instillation.

A significant inhibitory effect was observed with nicotine andepibatidine but not with DMPP. However, after increasing the number ofmice treated or not treated with DMPP to 15, we did observe asignificant inhibition compared to the non-treated group (FIG. 12).

Levels of TNF (a pro-inflammatory cytokine) are lower in thebroncho-alveolar lavage of DMPP-treated mice (FIG. 13) indicating thatthe down-regulation of inflammation may result from lower TNFconcentrations.

II—Asthma-like Inflammation

Example 6 In Vivo Asthma Model

Similar experiments were performed in ovalbumine-sensitized mice. DMPPallegedly decreases both the inflammatory response and thehyper-responsiveness to inhaled allergens and methacholine.

Groups of Balb/c mice were sensitized by intra-peritoneal injection of20 μg OVA protein (chicken egg albumin; Sigma-Aldrich) emulsified in 2mg aluminum hydroxide in PBS. After 4 weeks, challenge doses of 1.5%/50μl OVA were administered intranasally. The challenge was performed dailyfor 3 consecutive days and then the mice assessed for allergicinflammation of the lungs 24 h after the last aerosol exposure. Groupsof mice were treated with various concentrations of DMPP during thechallenge period. Broncho-alveolar lavage (BAL) was performed and thefluid centrifuged at 400 g to separate cells from liquid (FIGS. 14, 15,16 and 17).

The supernatants were used to determine lung IL-5 levels. The totalnumber of BAL cells and differential cell counts were evaluated (FIG.18).

The experiment was repeated with the optimal dose of DMPP to assess theairway responsiveness.

Measurement of AHR

Airway hyper-reactivity (AHR) in response to metacholine was measured inanesthetized, tracheotomized, ventilated mice using acomputer-controlled ventilator (FlexiVENT).

Increasing doses of metacholine ((0 mg/kg-32.5 mg/kg) were administeredthrough the jugular vein (FIGS. 19, 20).

Example 7 Effect of Agonist Treatment on mRNA Expression of IL-4

The effect of agonist treatment on mRNA expression of IL-4, a cytokinethat is well known to be involved in the development of asthma, was alsotested (53). Nicotine decreased LPS-induced IL-4 mRNA expression by upto 92% with 40 μM DMPP completely blocked IL-4 mRNA expression (FIG.21).

Example 8 Action of Various Agonists on Eosinophil Transmigration

To further investigate the effect of nicotinic agonists on thedown-regulation of inflammation in asthma, we tested the action ofvarious agonists on eosinophil transmigration.

Infiltration of eosinophils and other inflammatory cells into lungtissues is an important feature of asthma and the cause of airwayinflammation and hyper-responsiveness. The passage of inflammatory cellsfrom the circulation to the lung involves migration through the vascularendothelium, the basement membrane, and extra-cellular matrixcomponents. Inflammatory cells cross the basement membrane by producingproteinases. In these preliminary in vitro experiments, we investigatedthe effects of various nicotinic agonists on the migration of purifiedblood eosinophils through an artificial basement membrane (Matrigel®coated chemotaxis chamber). DMPP induces a dose-related inhibition ofeosinophils transmigration (FIG. 22), while this effect is reversed bythe antagonist mecamylamine (MEC) (FIG. 23). This inhibitory effect isfurther confirmed with other nicotinic agonists including nicotine,epibatidine and cytosine (FIG. 24). Results are expressed as apercentage of inhibition (agonists-treated cells) compared to thecontrol condition without the agonists.

These results suggest that nicotinic agonists down-regulate thesynthesis or activation of proteinases that degrade basement membranecomponents, thus inhibiting the migration of eosinophils into lungmucosa.

Example 9 Effect of Nicotinic Agonists on Collagen Production

Asthma is characterized by airway structural changes, includingsub-epithelial collagen deposition, that may be a cause for thechronicity of the disease. An imbalance between collagen synthesis andits degradation by fibroblasts may be involved in this process (56). Inpreliminary experiments, we investigated the effects of nicotinicagonists on collagen A1 synthesis produced by primary normalfibroblasts. Collagen A1 gene expression was evaluated by RT-PCR.

The results are expressed as percentage of gene expression in agoniststreated cells compared to non-treated cells.

DMPP inhibits collagen A1 gene expression in a dose-dependant manner(FIG. 25). Nicotine has a slight inhibitory effect at 1 and 10 μM,whereas higher concentrations had no effects (FIG. 26), probably due toa desensitization of the receptors. Lower doses may be necessary toachieve an inhibition and will be tested. The inhibitory effect is alsoobserved with epibatidine (FIG. 27).

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

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1. A method of treating pulmonary inflammation of an inflammatorydisease selected from the group consisting of asthma, interstitialpulmonary fibrosis (IPF), sarcoidosis, hypersensitivity pneumonitis(HP), chronic HP and bronchiolitis obliterans with organizingpneumonitis (BOOP) in an animal in need thereof having saidinflammation, comprising administering to said animal a nicotinicreceptors agonist selected from epibatidine.
 2. The method as defined inclaim 1, wherein said pulmonary inflammatory disease is asthma.
 3. Themethod as defined in claim 1, wherein said nicotinic receptors agonistis administered by oral, sublingual, intraperitoneal, intravenous,inhaled, intratracheal, intranasal, parenteral, topical, directinjection, infusion or intraocular administration.
 4. The method asdefined in claim 2, further comprising administering at least one ofbronchodilator, anti-inflammatory, antileukotriene or immunosuppressiveagent.
 5. The method as defined in claim 4, wherein said at least one ofbronchodilator, anti-inflammatory, antileukotriene or immunosuppressiveagent is administered by oral, sublingual, intraperitoneal, intravenous,inhaled, intratracheal, intranasal, parenteral, topical, directinjection, infusion or intraocular administration.
 6. A method oftreating bronchial or interstital inflammation from apulmonary-inflammatory disease selected from the group consisting ofasthma, interstitial pulmonary fibrosis (IPF), sarcoidosis,hypersensitivity pneumonitis (HP), chronic HP and bronchiolitisobliterans with organizing pneumonitis (BOOP) in an animal in needthereof having said inflammation, comprising administering to saidanimal a nicotinic receptors agonist selected from epibatidine.
 7. Themethod as defined in claim 6, wherein said pulmonary inflammatorydisease is asthma.